bioRxiv preprint doi: https://doi.org/10.1101/753525; this version posted September 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Understanding and eliminating the detrimental effect of endogenous thiamine 2 auxotrophy on metabolism of the oleaginous yeast Yarrowia lipolytica 3 4 Caleb Walker,1,§ Seunghyun Ryu,1,§ Richard J. Giannone,2 Sergio Garcia,1 Cong T. Trinh1,* 5 1Department of Chemical and Biomolecular Engineering, The University of Tennessee Knoxville, 6 TN 37996 7 2Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 8 9 §Equal contributions 10 *Corresponding author 11 Email: [email protected] 12 Tel: 865-974-8121 13 14 15 1 bioRxiv preprint doi: https://doi.org/10.1101/753525; this version posted September 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 16 ABSTRACT 17 Thiamine is an essential vitamin that functions as a cofactor for key enzymes in carbon and energy 18 metabolism for all living cells. While most plants, fungi and bacteria can synthesize thiamine de 19 novo, the oleaginous yeast, Yarrowia lipolytica, cannot. In this study, we used proteomics 20 together with physiological characterization to understand key metabolic processes influenced 21 and regulated by thiamine availability and identified the genetic basis of thiamine auxotrophy in 22 Y. lipolytica. Specifically, we found thiamine depletion results in decreased protein abundance 23 of the lipid biosynthesis pathways and energy metabolism (i.e., ATP synthase), attributing to the 24 negligible growth and poor sugar assimilation observed in our study. Using comparative 25 genomics, we identified the missing gene scTHI13, encoding the 4-amino-5-hydroxymethyl-2- 26 methylpyrimidine phosphate synthase for the de novo thiamine synthesis in Y. lipolytica, and 27 discovered an exceptional promoter, P3, that exhibits strong activation or tight repression by low 28 and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine- 29 regulated promoter (P3) to express the missing gene, we engineered the first thiamine- 30 prototrophic Y. lipolytica reported to date. By comparing this engineered strain to the wildtype, 31 we unveiled the tight relationship linking thiamine availability to lipid biosynthesis and 32 demonstrated enhanced lipid production with thiamine supplementation in the engineered 33 thiamine-prototrophic Y. lipolytica. 34 35 IMPORTANCE 36 Thiamine plays a crucial role as an essential cofactor for enzymes in carbon and energy 37 metabolism of all living cells. Thiamine deficiency has detrimental consequences on cellular 2 bioRxiv preprint doi: https://doi.org/10.1101/753525; this version posted September 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 38 health. Yarrowia lipolytica, a non-conventional oleaginous yeast with broad biotechnological 39 applications, is a native thiamine auxotroph, whose effects on cellular metabolism are not well 40 understood. Therefore, Y. lipolytica is an ideal eukaryotic host to study thiamine metabolism, 41 especially as mammalian cells are also thiamine-auxotrophic and thiamine deficiency is 42 implicated in several human diseases. This study elucidates the fundamentals of thiamine 43 deficiency on cellular metabolism of Y. lipolytica and identifies genes and novel thiamine- 44 regulated elements that eliminate thiamine auxotrophy in Y. lipolytica. Furthermore, discovery 45 of thiamine-regulated elements enables development of thiamine biosensors with useful 46 applications in synthetic biology and metabolic engineering. 47 48 Keywords: Yarrowia lipolytica; thiamine metabolism; thiamine auxotroph; thiamine prototroph; 49 thiamine-regulated promoters; lipid production; thiamine deficiency 50 51 52 3 bioRxiv preprint doi: https://doi.org/10.1101/753525; this version posted September 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 INTRODUCTION 54 Thiamine, or vitamin B1, was the first B vitamin discovered. Its activated form, thiamine 55 pyrophosphate (TPP), functions as a cofactor for key enzymes in carbon metabolism including 56 glycolysis, citrate cycle, pentose phosphate and branched chain amino acid pathways (Figure 1) 57 (1). TPP-dependent enzymes, including pyruvate dehydrogenase (PDH), α-ketoglutarate 58 dehydrogenase (KGDH), transketolase (TKL), branched chain α-ketoacid dehydrogenase (BCKDC) 59 and acetolactate synthase (AHAS), are essential for maintaining cell growth and preventing 60 metabolic stress (2, 3). Specifically, PDH links the glycolysis and citrate (Krebs) cycle by catalyzing 61 the conversion of pyruvate into acetyl-CoA (4)(5). KGDH, the rate-limiting step of the citrate 62 cycle, converts α-ketoglutarate to succinyl-CoA (6, 7). TKL is responsible for the last step of the 63 pentose phosphate pathway where it interconverts pentose sugars to glycolysis intermediates 64 (8). The pentose phosphate pathway is critical for production of ribose (i.e., RNA), precursor 65 metabolites for aromatic amino acid pathways, and reducing equivalents (i.e., NADPH) necessary 66 for maintaining redox balance and lipid synthesis. Hence, TKL activity is critical for RNA, protein, 67 and lipid production while preventing oxidative stress (9, 10). Meanwhile, AHAS and BCKDC are 68 responsible for the synthesis and degradation of branched chain amino acids (i.e., valine, leucine 69 and isoleucine), respectively (11)(12). 70 In mammals, thiamine deficiency affects the cardiovascular and nervous systems resulting 71 in tremors, muscle weakness, paralysis and even death (13)(14). Thiamine deficiency can occur 72 from inadequate intake, increased requirement, or impaired absorption of thiamine (15). 73 Biochemical consequences of thiamine deficiency result in failure to produce adenosine 74 triphosphate (ATP), increased acid production (i.e., lactic acid), decreased production of acetyl- 4 bioRxiv preprint doi: https://doi.org/10.1101/753525; this version posted September 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 75 compounds (i.e., acetylcholine), neurotransmitters (i.e., glutamate, aspartate, aminobutyric 76 acid), reduced nicotinamide adenosine dinucleotide phosphate (NADH), defective ribonucleic 77 acid (RNA) ribose synthesis, and failure to break down branched chain carboxylic acids (i.e., 78 leucine, valine, isoleucine)(16-18). 79 While mammals require nutritional supplementation of thiamine from dietary sources, 80 most bacteria, fungi and plants can synthesize thiamine endogenously. One of these exceptions 81 is the thiamine-auxotrophic oleaginous yeast, Yarrowia lipolytica, which has recently emerged as 82 an important industrial microbe with broad biotechnological applications due to its GRAS status 83 (19), metabolic capability (20-24), and robustness (25-27). Hence, Y. lipolytica is an ideal 84 eukaryotic host to study the fundamental effects of thiamine deficiency on cellular health. 85 Currently, it is not well understood why thiamine deficiency causes failure of thiamine 86 biosynthesis in Y. lipolytica and how this deficiency might directly affect other processes (e.g., 87 lipid biosynthesis, energy metabolism, etc.) beyond what is already known. 88 Interestingly, Y. lipolytica’s auxotrophy has been exploited for enhanced production of 89 organic acids (i.e., pyruvate and KGA) by reducing activities of PDH and KGDH under thiamine- 90 limited conditions (28, 29). Consequently, cell growth is negatively affected by thiamine 91 limitation and completely prevented when thiamine is depleted from the media. Not 92 surprisingly, the genes in thiamine metabolism are tightly regulated by thiamine concentrations 93 (30, 31). To this end, numerous thiamine-regulated promoters have been discovered in yeast 94 that enable control of genetic expression by adjusting thiamine concentration in the culture 95 media (32-34). However, endogenous thiamine-regulated promoters have not yet been 5 bioRxiv preprint doi: https://doi.org/10.1101/753525; this version posted September 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 96 discovered in Y. lipolytica. Furthermore, fundamental understanding of how thiamine deficiency 97 inhibits metabolism and hence growth of Y. lipolytica is still lacking. 98 In this study, we shed light on the effect of thiamine deficiency on cellular metabolism in 99 the thiamine auxotroph Y. lipolytica. We identified the missing